APOBEC3 enzymes are innate immune effectors that introduce mutations into viral genomes. These enzymes are cytidine deaminases which transform cytosine into uracil. They preferentially mutate cytidine preceded by thymidine making the 5'TC motif their favored target. Viruses have evolved different strategies to evade APOBEC3 restriction. Certain viruses actively encode viral proteins antagonizing the APOBEC3s, others passively face the APOBEC3 selection pressure thanks to a depleted genome for APOBEC3-targeted motifs. Hence, the APOBEC3s left on the genome of certain viruses an evolutionary footprint. The aim of our study is the identification of these viruses having a genome shaped by the APOBEC3s. We analyzed the genome of 33,400 human viruses for the depletion of APO-BEC3-favored motifs. We demonstrate that the APOBEC3 selection pressure impacts at least 22% of all currently annotated human viral species. The papillomaviridae and polyomaviridae are the most intensively footprinted families; evidencing a selection pressure acting genome-wide and on both strands. Members of the parvoviridae family are differentially targeted in term of both magnitude and localization of the footprint. Interestingly, a massive APOBEC3 footprint is present on both strands of the B19 erythroparvovirus; making this viral genome one of the most cleaned sequences for APOBEC3-favored motifs. We also identified the endemic coronaviridae as significantly footprinted. Interestingly, no such footprint has been detected on the zoonotic MERS-CoV, SARS-CoV-1 and SARS-CoV-2 coronaviruses. In addition to viruses that are footprinted genome-wide, certain viruses are footprinted only on very short sections of their genome. That is the case for the gamma-herpesviridae and adenoviridae where the footprint is localized on the lytic origins of replication. A mild footprint can also be detected on the negative strand of the reverse transcribing HIV-1, HIV-2, HTLV-1 and HBV viruses. Together, our data illustrate the extent of the APOBEC3 selection pressure on the human viruses and identify new putatively APOBEC3-targeted viruses.

The emergence of the SARS-CoV-2 virus and the exponential growth of COVID-19 cases have created a major crisis for public health systems. The critical identification of contagious asymptomatic carriers requires the isolation of viral nucleic acids, reverse transcription, and amplification by PCR. However, the shortage of specific proprietary reagents or the lack of automated platforms have seriously hampered diagnostic throughput in many countries. Here, we provide a procedure for SARS-CoV-2 detection for diagnostic purposes from clinical samples in the setting of a basic research molecular biology lab. The procedure details the necessary steps for daily analysis of up to 500 clinical samples with a team composed of 12 experienced researchers. The protocol has been designed to rely on widely available reagents and devices, to cope with heterogeneous clinical specimens, to guarantee nucleic acid extraction from very scarce biological material, and to minimize the rate of false-negative results.

Modelling cell infection in-a-dish can represent a useful tool to understand the susceptibility of different cell types towards severe acute respiratory coronavirus-2 (SARS-CoV-2) and to decipher its neurotropism. In this perspective, retinoic acid (RA)-differentiated neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2) and glioblastoma cell lines, U-87 MG and U-373 MG, were infected with a SARS-CoV-2 strain, at various multiplicity-of-infection (MOI). We first demonstrated that the common entry genes – needed for invading epithelial cells – were expressed. RA-differentiation induced an upregulation of ace2 and tmprss2 gene expression while inducing downregulation of ctsb and ctsl . Using in situ hybridization and confocal analysis, SARS-CoV-2 gene S RNA was detected intracellularly at MOI 5.0, and localized in both soma and neuritic-like or glial-like processes. The infection was confirmed by quantification of viral gene E RNA and showed a dose-dependency, with few infected cells at MOI 0.1. After 24 h of infection, no cytopathic effect was observed in SH-SY5Y abilities to maintain neuritic processes or in U-373 MG for the uptake of glutamate. Unlike the permissive Vero E6 cells, no significant apoptosis death was detected following SARS-CoV-2 infection of neuroblastoma or glioblastoma cells. This study demonstrates the susceptibility of neuronal- and glial-like cell lines towards SARS-CoV-2 infection at high MOIs. Once inside the cells, the virus does not seem to rapidly replicate nor exert major cytopathic effect. Overall, our results strengthen the idea that SARS-CoV-2 has a tropism for nervous cells that express commonly described entry genes.

Human adenoviruses (HAdVs) are a large family of DNA viruses counting more than 100 genotypes divided into seven species (A–G) and inducing respiratory tract infections, gastroenteritis and conjunctivitis. Genetically modified adenoviruses are also used as vaccines, gene therapies and anti-cancer treatments. The APOBEC3s are a family of cytidine deaminases that restrict viruses by introducing mutations in their genomes. Viruses developed different strategies to cope with the APOBEC3 selection pressure but nothing is known on the interplay between the APOBEC3s and the HAdVs. In this study, we focused on three HAdV strains: the B3 and C2 strains as they are very frequent and the A12 strain, less common but oncogenic in animal models. We demonstrated that the three HAdV strains induce a similar APOBEC3B upregulation at the transcriptional level. At the protein level however, the APOBEC3B is abundantly expressed during the HAdV-A12 and -C2 infection and shows a nuclear distribution. On the contrary, APOBEC3B is barely detectable in HAdV-B3-infected cells. APOBEC3B deaminase activity is detected in total protein extracts upon HAdV-A12 and -C2 infection. Bioinformatic analysis demonstrate that the HAdV-A12 genome bears a stronger APOBEC3 evolutionary footprint than the HAdV-C2 and HAdV-B3 genomes. Our results show that HAdV infection triggers the transcriptional upregulation of the antiviral innate effector APOBEC3B. The discrepancies between the APOBEC3B mRNA and protein levels might reflect the ability of some HAdV strains to antagonize the APOBEC3B protein. These findings point toward an involvement of APOBEC3B in HAdVs restriction and evolution. IMPORTANCE The APOBEC3 family of cytosine deaminases has important roles in antiviral innate immunity and cancer. Notably, APOBEC3A and/or APOBEC3B are actively upregulated by several DNA tumor viruses and contribute to transformation by introducing mutations in the cellular genome. Human adenoviruses (HAdVs) are a large family of DNA viruses causing generally asymptomatic infections in immunocompetent adults. HAdVs encode several oncogenes and some HAdV strains like HAdV-A12 induce tumors in hamsters and mice. Here, we show that HAdV infection specifically promotes the expression of the APOBEC3B gene. We report that infection with the A12 strain induces a strong expression of an enzymatically active APOBEC3B protein in bronchial epithelial cells. We provide bioinformatic evidences that HAdVs’ genomes and notably the A12 genome are under APOBEC3 selection pressure. Thus, APOBEC3B might contribute to adenoviral restriction, diversification and oncogenic potential of particular strains.

Human adenoviruses (HAdVs) are a large family of DNA viruses counting more than a hundred strains divided into seven species (A to G). HAdVs induce respiratory tract infections, gastroenteritis and conjunctivitis. APOBEC3B is a cytidine deaminase that restricts several DNA viruses. APOBEC3B is also implicated in numerous cancers where it is responsible for the introduction of clustered mutations into the cellular genome. In this study, we demonstrate that APOBEC3B is an adenovirus restriction factor acting through a deaminase-dependent mechanism. APOBEC3B introduces C-to-T clustered mutations into the adenovirus genome. APOBEC3B reduces the propagation of adenoviruses by limiting viral genome replication, progression to late phase, and production of infectious virions. APOBEC3B restriction efficiency varies between adenoviral strains, the A12 strain being more sensitive to APOBEC3B than the B3 or C2 strains. In A12-infected cells, APOBEC3B clusters in the viral replication centers. Importantly, we show that adenovirus infection leads to a reduction of the quantity and/or enzymatic activity of the APOBEC3B protein depending on the strains. The A12 strain seems less able to resist APOBEC3B than the B3 or C2 strains, a characteristic which could explain the strong depletion of the APOBEC3-targeted motifs in the A12 genome. These findings suggest that adenoviruses evolved different mechanisms to antagonize APOBEC3B. Elucidating these mechanisms could benefit the design of cancer treatments. This study also identifies adenoviruses as triggers of the APOBEC3B-mediated innate response. The involvement of certain adenoviral strains in the genesis of the APOBEC3 mutational signature observed in tumors deserves further study.